The invention relates to fusion polypeptides. It concerns fusion polypeptides comprising an IgE-binding domain and a human serum albumin (HSA) component and salts thereof. It also concerns polynucleotides and physiologically functional equivalent polypeptides which are intermediates in the preparation of such fusion polypeptides; appropriate recombinant expression vectors therefor, corresponding procaryotic and eucaryotic expression systems, and processes for synthesizing the fusion polypeptides.
The interaction between immunoglobulin E (IgE) and its receptors has an established role in the defense against parasitic infections in humans (M. Capron and A. Capron. Science 264 [1994] 1876-1877). In industrialized countries, however, with improved hygienic conditions, the encounter with parasites is less frequent than the disturbance of the IgE network by over-production of IgE in response to environmental allergens. resulting in allergies and other IgE- or IgE-receptor- mediated disease states.
IgE is the primary antibody involved in initiation of an immediate allergic response and is a major participant in the maintenance of the late phase response. IgE is synthesized in B lymphocytes and exerts its effects after binding to the high affinity receptor for IgE, i.e. Fcxcex5RI, which is found on the surface of allergy effector cells such as mast cells, basophils and eosinophils. IgE also exerts inducer functions through binding to its receptor on antigen presenting cells such as Langerhans cells, B cells and monocytes (G. C. Mudde et al., Allergy 50 [1995] 193-199).
An allergic response or condition is manifested when IgE molecules bind to the surface of allergy effector cells via the IgE receptor, Fcxcex5RI, and become cross-linked by allergen, thereby effecting signalling to initiate degranulation of cytoplasmic granules in the cells, with accompanying release of mediators of allergy such as histamine, serotonin, prostaglandins and cytokines, and consequent local tissue edema and influx of inflammatory cells. An alternate means of stimulating allergy and associated conditions is the interaction of the IgE receptor, Fcxcex5RI, with circulating autoantibodies against Fcxcex5RI.
Fcxcex5RI typically exists as a tetramer comprising an xcex1-, a xcex2- and two xcex3-chains (i.e. xe2x80x9csubunitsxe2x80x9d), although on monocytes and Langerhans cells, the xcex2-subunit is absent.
The IgE binding site of Fcxcex5RI has been shown to be contained entirely within its xcex1-subunit (referred to as Fcxcex5RIxcex1) (J. Hakimi et al., J. Biol. Chem. 265 [1990] 22079-22081; U. Blank et al., J. Biol Chem. 266 [1991] 2639-2646). Recombinant xe2x80x9cknockoutxe2x80x9d mice genetically deleted for the entire xcex1-subunit have been found to be unable to mount an allergic response to allergen challenge (D. Dombrowicz et al., Cell 75 [1993] 969-976).
Fcxcex5RIxcex1 is a heavily glycosylated polypeptide of molecular weight about 60 kD, comprising a hydrophobic transmembrane domain as well as hydrophilic extracellular (xe2x80x9cecto-xe2x80x9d) and cytoplasmic domains which are exposed to the outer surface of the cell. The IgE binding capability of Fcxcex5RIxcex1 has been been further localized to its extracellular portion (J. Hakimi et al. [11990], supra; Leder et al., U.S. Pat. No. 4,962,035). It is possible to produce a soluble, secretable molecule by excising the transmembrane portion and sequences downstream therefrom (C. Ra et al., Int. Immunol. 5 [1993] 47-54); and the resulting truncation, consisting essentially of the human Fcxcex5RIxcex1 extracellular domain, has IgE-binding activity in vitro and in vivo (M. Haak-Frendscho et al., Immunol. 151 [1993] 351-358: amino acid residues 1-204 of Fcxcex5RIxcex1 fused to a truncated IgGl H chain C region; C. Ra. et al. [1993] supra: residues 1-172 of Fcxcex5RIxcex1 therein, corresponding to residues 26-197 of SEQ. ID. NO. 1 hereof). Structural features of this fragment include two potential disulfide bridges and seven potential glycosylation sites (M. Haak-Frendscho et al. [1993], supra).
Therefore, truncations of Fcxcex5RIxcex1 consisting essentially of the extracellular domain can potentially be administered therapeutically to a mammal to bind serum IgE in order to prevent its binding to its high affinity receptor on allergy effector cells, and also for suppressing de novo IgE biosynthesis in human lymphocytes (Y. Yanagihara et al., J. Clin. Invest. 94 [1994] 2162-2165).
However, effective use of an IgE-binding polypeptide such as the extracellular domain of Fcxcex5cRIxcex1 for systemic treatment of IgE- or IgE receptor- mediated allergic disorders in mammals has been hindered by its extreme transience in vivo due to rapid clearance from circulating plasma. Considering that IgE- or IgE receptor- mediated diseases account for 10-20% of physician-patient contact, an effective treatment would constitute a significant benefit to patients suffering from such conditions and an important advance in the clinical treatment of IgE- and IgE receptor- mediated disorders such as allergy and allergy-related conditions.
It would therefore be beneficial to obtain an IgE-binding polypeptide having prolonged effective serum life and thus improved clinical utility in the treatment of allergy, and in particular the systemic treatment of atopic dermatitis, atopic asthma and chronic urticaria, as well as improved activity in an efficient, cost-effective manner.
It has now been found that by fusing an IgE-binding domain to a human serum albumin (HSA) component, fusion polypeptides can be obtained having extended serum half-life relative to the IgE-binding domain alone, without loss of IgE-binding activity, resulting in IgE-binding polypeptides indicated for use in the systemic treatment of allergy and other IgE-mediated disorders. Systemically administered IgE-binding polypeptide will bind to serum IgE as well as to circulating auto-antibodies against the IgE receptor, Fcxcex5RIxcex1, preventing them from binding to cell-bound Fcxcex5RIxcex1, and thus preventing and/or inhibiting an allergic reaction and its associated manifestations.
It has further been found that significant improvements in IgE-binding activity can be obtained by using fusion polypeptides of the invention comprising more than one IgE-binding domain per molecule. For example, a xe2x80x9cdimerxe2x80x9d molecule of the invention has been found to have significantly increased IgE-binding activity relative to a xe2x80x9cmonomerxe2x80x9d of the invention.
The term xe2x80x9cdimerxe2x80x9d herein refers to a fusion polypeptide of the invention possessing two IgE-binding domains. The term xe2x80x9cmonomerxe2x80x9d refers to a fusion polypeptide of the invention possessing one IgE binding domain. The monomer or dimer may be constructed of a single or multiple HSA components. For example, a monomeric fusion polypeptide of the invention may comprise an IgE-binding domain fused at either its amino or carboxy terminus to an HSA component. Alternatively, the monomer may comprise an IgE-binding domain fused at both of its termini to HSA components. A dimeric fusion polypeptide according to the invention may comprise, for example, two IgE-binding domains fused via the carboxy terminus of one and the amino terminus of the other to an intervening HSA component. Alternatively, the dimer may comprise, in addition to its two IgE-binding domains, multiple HSA components.
It has further been found that a dimer molecule of the invention possesses unexpectedly favorable activity.
The invention therefore is directed to fusion polypeptides and salts thereof comprising at least one IgE-binding domain fused to at least one HSA component; preferably. to monomeric fusion polypeptides, wherein a single IgE-binding domain is fused to one or more HSA components, or to multimeric fusion polypeptides, wherein two or more IE-binding domains are fused to at least one HSA component; more preferably, dimers possessing two IgE-binding domains and at least one HSA component.
The invention is further directed to polynucleotide intermediates therefor.
The term xe2x80x9cfusedxe2x80x9d or xe2x80x9cfusionxe2x80x9d herein refers to polypeptides in which:
(i) a given functional domain (i.e. an IgE-binding domain) is bound at its carboxy terminus by a covalent (preferably, peptide, i.e. amide) bond either to the amino terminus of another functional domain (i.e. an HSA component) or to a linker peptide which itself is bound by a covalent (preferably, peptide) bond to the amino terminus of the other functional domain; and/or,
(ii) a given functional domain (i.e. an IgE-binding domain) is bound at its amino terminus by a covalent (preferably, peptide, i.e. amide) bond either to the carboxy terminus of another functional domain (i.e. an HSA component) or to a linker peptide which itself is bound by a covalent (preferably, peptide) bond to the carboxy terminus of the other functional domain.
Similarly, xe2x80x9cfusedxe2x80x9d when used in connection with the polynucleotide intermediates of the invention means that the 3xe2x80x2- [or 5xe2x80x2-] terminus of a nucleotide sequence encoding a first functional domain is bound to the respective 5xe2x80x2-[or 3xe2x80x2-] terminus of a nucleotide sequence encoding a second functional domain, either by a covalent bond or indirectly via a nucleotide linker which itself is covalently bound preferably at its termini to the first functional domain-encoding polynucleotide and the second functional domain-encoding polynucleotide.
Preferably, the fusion polypeptide or its salt is a monomer or a dimer; when it is a dimer it comprises two IgE-binding domains fused to an HSA component, such as a first IgE-binding domain fused at its carboxy terminus to the amino terminus of an HSA component, and a second IgE-binding domain fused at its amino terminus to the carboxy terminus of that HSA component.
The fusion polypeptides of the invention may also comprise further functional domains in addition to IgE-binding domains and HSA components, e.g. full length or truncated (e.g. soluble, extracellular fragments of) human proteins, such as cytokines or the amino-terminal fragment of urokinase. These additional functional domains may themselves serve as linker peptides, for example, for joining an IgE-binding domain to another IgE-binding domain or to an HSA component; alternatively, they may be located elsewhere in the fusion molecule, e.g. at the amino or carboxy terminus thereof.
Examples of fusion polypeptides of the invention may be represented by the following formulas:
R1-L-R2xe2x80x83xe2x80x83I.
R2-L-R1xe2x80x83xe2x80x83II.
R1-L-R2-L-R1xe2x80x83xe2x80x83III.
R1-L-R1-L-R2xe2x80x83xe2x80x83IV.
R2-L-R1-L-R1xe2x80x83xe2x80x83V.
wherein
R1 is the amino acid sequence of an IgE-binding domain,
R2 is the amino acid sequence of an HSA component,
each L is independently a covalent (preferably, peptide) bond, or is a peptide linker which is
bound by a covalent (preferably, peptide) bond to a terminus of R1 and/or R2, whereby the above molecule fragments are read directionally, i.e. with the left side
corresponding to the amino terminus and the right side to the carboxy terminus of the molecule.
It has been found that IgE binding is at high levels when the fusion polypeptide is led by an IgE-binding domain; that is, when the IgE binding domain constitutes the N-terminal portion of the mature fusion protein (such as in compounds of formulae I, III and IV, above). A particularly preferred embodiment of the invention comprises a dimer of formula III above, i.e. wherein the protein region which leads expression is an IgE-binding domain, fused at its carboxy terminus to the amino terminus of an HSA component, which in turn is itself fused at its carboxy terminus to the amino terminus of a second IgE-binding domain. When there is more than one R1 moiety, or more than one R2 moiety, or more than one L moiety, these moieties can be, but are not necessarily, identical.
Preferably the fusion polypeptides of the invention comprise at least one soluble, secretable mammalian IgE-binding domain fused to at least one HSA component, and pharmaceutically acceptable salts thereof.
The invention is further directed to a method for the prevention and/or treatment of IgE- or IgE-receptor- mediated disorders, and in particular, allergic reactions such as atopic dermatitis, atopic asthma and chronic urticaria, comprising the administration of the fusion polypeptides of the invention or pharmaceutically acceptable salts thereof to a subject in need of such treatment; and to pharmaceutical compositions comprising the fusion polypeptides of the invention or pharmaceutically acceptable salts thereof, together with at least one pharmaceutically acceptable carrier or diluent.
The invention is further directed to physiologically functional equivalents of the fusion polypeptides of the invention which are intermediates in the synthesis of the polypeptides; to polynucleotides which are intermediates in the preparation of the fusion polypeptides or physiologically functional equivalents thereof as defined above; to oligonucleotide intermediates to be used to construct them; to the peptides encoded by such oligonucleotides; to recombinant vectors for expressing the fusion molecules; to procaryotic or eucaryotic (especially, mammalian) expression systems; and to processes for preparing the fusion polypeptides or physiologically functional equivalents thereof using such expression systems.
SEQ. ID. NO. 1: Amino acid sequence of dominant form of full length native human Fcxcex5RIxcex1, including signal sequence.
The term xe2x80x9cpre-IgERxe2x80x9d refers to residues Met1-Leu204 of SEQ. ID. NO. 1. The term xe2x80x9cIgERxe2x80x9d refers to the mature form of pre-IgER and constitutes residues Val26-Leu204 of SEQ. ID. NO. 1 (i.e. the extracellular domain of Fcxcex5RIxcex1).
SEQ. ID. NO. 2: Amino acid sequence of dominant form of native prepro-HSA (referred to herein as xe2x80x9cprepro HSA Ixe2x80x9d), comprising residues Met1-Leu609.
The dominant form of the mature native protein (referred to herein as xe2x80x9cHSA Ixe2x80x9d) is represented by residues Asp25-Leu609 of SEQ. ID. NO. 2.
The term xe2x80x9cprepro-HSA IIxe2x80x9d represents a truncation of the native sequence by one amino acid (Leu609) at the carboxy terminus, and therefore refers to residues Met1-Gly608 of SEQ. ID. NO. 2.
The mature form of prepro-HSA II, referred to herein as xe2x80x9cHSA IIxe2x80x9d, is represented by residues Asp25-Gly608 of SEQ. ID. NO. 2.
SEQ. ID. NO. 3: Amino acid sequence encoded by the EcoRI fragment of plasmid R-H-R/SK#50 prepared in Example 5, comprising: xe2x80x9cpre-IgERxe2x80x9d sequence at residues 1-204; linker AlaSer(Gly)4Ser (referred to hereinafter as xe2x80x9cL1xe2x80x9d) at residues 205-211; HSA II sequence at residues 212-795; linker (Gly)3Ser (referred to hereinafter as xe2x80x9cL2xe2x80x9d) at residues 796-799; and the xe2x80x9cIgERxe2x80x9d sequence at residues 800-978.
A mature dimeric fusion polypeptide of the invention, referred to herein as xe2x80x9cIgER-L1-HSA II-L2-IgERxe2x80x9d or, alternatively, as xe2x80x9cIgER-HSA-IgER Dimerxe2x80x9d, expressed from CHO cells in the manner described in Example 7, has the amino acid sequence Val26-Leu978 of SEQ. ID. NO. 3.
SEQ. ID. NO. 4: Nucleotide sequence of the EcoRI fragment of plasmid R-H-RISK #50 of Example 5, comprising: a polynucleotide sequence encoding xe2x80x9cpre-IgERxe2x80x9d at positions 10-621; an oligonucleotide encoding L1 at positions 622-642; a polynucleotide encoding HSA II at positions 643-2394; an oligonucleotide encoding L2 at positions 2395-2406; and a polynucleotide encoding xe2x80x9cIgERxe2x80x9d at positions 2407-2943; with 2 stop codons at positions 2944-2949.
Restriction sites at the ends of the coding fragments and in the linker regions are at positions 1-6; 622-627; 637-642; 2387-2393; 2401-2406; and 2950-2955. A Kozak sequence is at nucleotide positions 7-9.
Point mutations differing from the consensus HSA nucleotide sequence are at positions 804, 1239; 1290; 1446, 1815, 2064 and 2079. Because the point mutations are in the wobble position, they do not affect the amino acid sequence.
Seq. ID. NO. 5: Nucleotide sequence of the dominant form of native prepro-HSA corresponding to FIG. 12 and to the amino acid sequence of SEQ. ID. NO. 2.
SEQ. ID. NO. 6: Nucleotide sequence of the dominant form of full length native human Fcxcex5RIxcex1 including signal sequence, corresponding to FIG. 13 and to the amino acid sequence of SEQ. ID. NO. 1.
SEQ. ID. NO. 7: HSA, IgER and mutagenic sequencing oligonucleotides